【作者】 张帆；

【导师】 刘向东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2011， 硕士

【摘要】 南京地区的棉蚜种群在寄主利用和迁飞能力上存在明显的分化。在寄主利用上可分为棉花型和瓜型,在迁飞能力上可分为迁飞型和滞留型。但是,棉蚜种群的寄主型和迁飞型产生的分子机理,尚无定论。本文对南京地区迁飞型和寄主型棉蚜的不同品系的交配行为、mtDNA的COⅠ和COⅡ基因以及内共生菌Buchnera相关基因的序列变异、以及Buchnera共生菌的数量等方面进行了研究,以期探讨寄主型和迁飞型棉蚜产生的分子基础,取得了以下主要结果：(1)对南京地区棉花型、瓜型、迁飞型和滞留型棉蚜,在低温短光照条件(18℃,L:D=8:16)下进行性蚜诱导,比较分析了棉花型和瓜型之间,以及迁飞型和滞留型之间性蚜的交配行为。结果表明,迁飞型棉蚜在诱导条件下容易产生有性世代,产生雄性蚜和雌性蚜的时间要比滞留型棉蚜早5天左右。迁飞型和滞留型棉蚜的性蚜间可以发生交配行为,但同型交配的效率要高于异型交配,具体表现为迁飞型自交的寻偶时间显著地短于正交(M(?)×S(?))和反交(S(?)×M(?)),但与滞留型自交时的时间无显著差异。迁飞型M(?)和滞留型S(?)杂交时的交配持续时间显著长于两者自交时的时间。棉花型和瓜型的性蚜间可以发生交配行为,且杂交时的寻偶时间与交配持续时间在正交(Cu(?)×Co(?))和反交(Co(?)×Cu(?))中均无显著差异。(2)测定了棉蚜不同品系mtDNA基因序列上的变异,结果发现,室内孤雌饲养七年的棉花型和瓜型棉蚜,以及从棉花和黄瓜田间采集的自然棉蚜均未发生COⅠ序列上的变异,但在2008年和2010年选育出的迁飞型棉蚜种群中均检测到了2个COⅠ序列的突变位点,在2008年的滞留型棉蚜的一个个体中检测到1个COⅠ序列的突变位点,但在2010年的滞留型棉蚜中没有再次检测到。在mtDNA水平上,南京地区的棉蚜存在四种单倍型H1,H2,H3和H4,并且棉花型、瓜型、迁飞型和滞留型棉蚜还均共享着同一单倍型H4。系统发育分析显示,南京地区棉蚜种群可划分为三个支系,支系一由COⅠ序列没有发生突变的个体所组成,其夏寄主为棉花、瓜类或木槿,该支系个体占绝大多数；支系二和支系三的COⅠ序列均发生了一个碱基的突变,并且均由冬寄主上营异寄主生活史的迁飞型所组成,且个体数量相对较少。棉花型、瓜型、迁飞型和滞留型棉蚜在Leu-tRNA/COⅠⅠ序列上未发生变异。发生了COⅠ位点突变的与没有突变的迁飞型棉蚜在净增殖率R0、平均寿命T及内禀增长率rm上均没表现出显著差异,COⅠ序列的变异与棉蚜的生态适应性没有直接相关性。(3)选取迁飞型棉蚜发生COⅠ变异的H3单倍型M-H3、未发生变异的H4单倍型M-H4、滞留型棉蚜S、室外棉花上棉蚜Cow、室外黄瓜上棉蚜Cuw以及室内棉花型棉蚜Co和瓜型棉蚜Cu等7个品系,测定了其内共生菌Buchnera的1euA基因分化程度。结果显示,瓜型棉蚜的两个群体(Cu和Cuw)发生了该基因序列上的一个碱基的突变,而棉花型、迁飞型和滞留型间没有突变发生。系统发育分析显示,瓜型棉蚜的两个群体处于同一分支,其它品系为另一分支,棉花型和瓜型棉蚜寄主利用能力的分化与其内共生菌有关。(4)研究了以上7个品系棉蚜Buchnera的trpEG基因分化程度及Buchnera在棉蚜不同品系内的含量。结果显示,在黄瓜上取食的瓜型棉蚜与在棉花上取食的棉花型、迁飞型和滞留型棉蚜在trpEG上有着两个碱基的差异。系统发育分析显示,在黄瓜上生活的棉蚜聚为一支,其它生活在棉花上的品系聚为一支。对Buchnera进行定量PCR发现,瓜型棉蚜体内Buchnera菌含量显著高于其它品系。由此说明原生共生菌与棉蚜的寄主专化型的形成有一定关系。更多还原

【Abstract】 There are obvious differentiations in host use and migratory capacity of cotton aphid, Aphis gossypii Glover in Nanjing. The cotton aphid can be classified as cotton-and cucurbit-specialized strains in using host plant, and they can also be classified as migratory and sedentary strains in migratory capacity. But the molecular mechanisms of host-specialization and migration are still vague. The behaviour of the copulation, mutation in COⅠ/COⅡand Buchnera genes sequences, and the content of endosymbionts Buchnera in migratory and host-specialized biotypes of Aphis gossypii Glover in Nanjing were studied in this paper, in order to explore the molecular background of the formation of host-specialization and migratory biotypes. The results were showed as follows:(1) The sexual generation of the cotton-(Co), cucurbits-(Cu) specialized, migratory (M) and sedentary (S) strains of cotton aphid were induced under the low temperature (18℃) and short photoperiod (L:D=8:16), and the mating behavior between different strains was also compared. The results showed that migratory strain was prone to give birth to sexual generation. The date generated male and female aphids was earlier about 5 days to the sedentary strain. The durations of finding spouse of M♀×M(?) was significantly shorter than those of the copulation M♀×S(?) and S♀xM(?) but there was not significantly different between M♀×M(?) and S♀×S(?). The durations of mating of M♀×S(?) was significantly longer than that of the M♀×M(?) and S♀×S(?),but there was no significant difference between M♀×S(?) and S♀×M(?).The assortative mating behavior was found between migratory and sedentary strains. The durations of finding spouse and mating were no significant difference between Cu♀×Co(?) and Co♀×Cu(?). The mating behavior of different strains of cotton aphid revealed that mating behavior could happen between host-specialized aphids, and between migratory and sedentary strains, and there was no segregation at sexual generation.(2) Sequence variations in the mtDNA genes COI and COII among different strains of cotton aphid were determined. The results indicated that there was no COI sequence mutation in cotton- and cucurbits-specialized strains even if they had been reared seven years in laboratory on cotton and cucumber plant, respectively, or were collected from natural field of cotton and cucumber, But there were two bases mutations in migratory strains of 2008 and 2010 population, and one base mutation in sedentary strain of 2008. However, the mutation in sedentary strain was not detected again in 2010 population. There were four haplotypes (H1, H2, H3, H4) of cotton aphid in Nanjing at mtDNA level, and all the cotton-and cucurbit-specialized strains, migratory and sedentary strains shared the same haplotype H4. Phylogenetic analysis showed that cotton aphid population in Nanjing could be divided into three clades. Clade 1 was dominant and no any mutation in COⅠand those aphids fed on the hibiscus, cotton and cucurbits host plants in summer. The clade 2 and clade 3 were comprised of migratory strain, and contented some mutations in COⅠgene. Not any mutations in Leu-tRNA/COⅡsequence were found in the above strains. Mutation in COⅠgene sequence did not affect the fecundity and fitness of the aphids.(3) Seven strains of cotton aphid, M-H3 (haplotype H3), M-H4 (haplotype H4), S (sedentary strain), Cuw (just collected from cucumber field), Cow (just collected from cotton field), Co (cotton-specialized) and Cu (cucurbit-specialized) were determined in the differentiation of the endosymbionts Buchnera’s leuA gene. The results showed that the two groups of cucurbit-specialized strains (Cu and Cuw) contented one base mutation in leuA gene sequence, while the cotton strain, migration and sedentary strain had no mutation. Phylogenetic analysis showed that the two groups of cucurbit-specialized strains (Cu and Cuw) were in the same branch, and the other strains were in the other branch. Cotton- and cucurbit-specialization of cotton aphid may be related to their endosymbionts.(4) Buchnera trpEG genetic differentiation and Buchnera quantity in the above seven strains of cotton aphid were studied. The results showed that two bases mutations were found in the trpEG sequence of the cucurbit-specialized strain and the cotton aphid collected just from cucumber. No mutation in cotton-specialized strain, migration, sedentary strain was detected. Phylogenetic analysis showed that the aphids living on cucumber were clustered firstly, and the other cluster tree was aphids living on cotton. Quantitative PCR of Buchnera discovered that Buchnera in cucurbit-specialized strains was significantly higher than that of the other strains. The results revealed that the primary symbiots of cotton aphid was close related to the host-specialized biotypes.更多还原